Reagents and methods for detecting pnh type ii white blood cells and their identification as risk factors for thrombotic disorders

ABSTRACT

The disclosure relates to methods for detecting PNH Type II cell populations in biological samples as well as methods for determining whether a patient is at an increased risk for developing thrombocytopenia or thrombosis based on the percentage of PNH Type II cells in the patient&#39;s blood. The disclosure also features reagents and conjugates for use in the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application Ser. No. 61/280,897, filed Nov. 9, 2009, and entitled“Reagents and methods for detecting PNH type II cells”, the entirecontents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 5, 2010, is namedALXN150WO1.txt, and is 26,000 bytes in size.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, debilitatingdisease that is characterized by, among other things, abnormalhematopoiesis, complement-mediated intravascular hemolysis, and apropensity for thrombosis. See, e.g., Rosse and Nishimura (2003) Int JHematol 77(2):121-124 and Brodsky (2008) Blood Rev 22(2):65-74. PNH iscaused by a somatic mutation in the X-linked phosphatidylinositol glycancomplementation class A (PIGA) gene, which encodes an enzyme that isnecessary for the initial step of glycosylphosphatidylinositol (GPI)anchor biosynthesis. See Miyata et al. (1993) Science 259:1318-1320 andBessler et al. (1994) EMBO J 13:110-117. GPI anchors attach a number ofproteins to the surface of hematopoietic cells. These so calledGPI-anchored proteins include, among others, complement regulatoryproteins such as CD55 (DAF) and CD59. Depending on the type of mutationthat befalls the PIGA gene, a partial or complete loss of GPI anchorbiosynthesis can result, which corresponds to a partial or complete lossin the presence of GPI-anchored proteins (e.g., GPI-anchored CD55 andCD59) on the cell surface. See Rosse (1997) Medicine 76:63-93. Thepartial or complete absence of complement regulatory proteins on thesurface of red blood cells (RBCs) results in the heightened sensitivityof these cells for complement-mediated lysis and associated symptoms ofPNH in afflicted patients. See Nicholson-Weller et al. (1983) Proc NatlAcad Sci USA 80:5066-5070 and Yamashina et al. (1990) N Engl J Med323:1184-1189.

Traditionally, diagnosis of PNH and monitoring of PNH patients involvedanalysis of CD55 and CD59 expression on the surface of RBCs andgranulocytes using flow cytometry. Sutherland et al. (2009) Am J ClinPathol 132:564-572. More recently developed diagnostic methods for PNHhave employed a recombinant, non-lytic form of the bacterial proteinaerolysin, which binds to GPI-anchors on the surface of hematopoieticcells. See U.S. Pat. No. 6,593,095 issued to Buckley and Brodsky. Bothtraditional and new methods have allowed medical practitioners toclassify RBCs or white blood cells from PNH patients into one of threegroups: Type I cells having normal or nearly normal cell-surfaceexpression of GPI-anchored proteins; PNH Type III cells, which have nilor completely absent cell-surface expression of GPI-anchored proteins;and PNH Type II cells having an intermediate level of cell-surfaceexpression of GPI-anchored proteins. Brodsky et al. (2000) Am J ClinPathol 114:459-466. The characterization of Type II cells among whiteblood cell lineages has not been performed due to the difficulty indistinguishing these cells from normal Type I white blood cells.

SUMMARY

The disclosure is based, at least in part, on the discovery by theinventors that patients having a PNH Type II white blood cell populationof at least 1.2% or a PNH Type II red blood cell population of at least0.02% are more likely to have thrombocytopenia as compared to patientswho do not have PNH Type II cell populations or who have PNH Type IIcell populations that are smaller than 1.2% or 0.02% for white and redblood cells, respectively. Patients with thrombocytopenia resulting fromplatelet destruction are much more likely to develop thrombosis, andamong PNH patients, thrombosis is the leading cause of death.Accordingly, the disclosure provides methods for determining whether apatient is at an increased risk for thrombocytopenia and/or thrombosisbased on the relative population of PNH Type II cells in the patient.Identification of the nexus between PNH Type II cells andthrombocytopenia was aided, in part, by the development of improvedmethods for detecting PNH Type II cells.

Thus, the disclosure also provides reagents and methods useful fordetecting PNH Type II cells (e.g., Type II white blood cells and/or TypeII red blood cells) in, e.g., biological samples from patients. Thedisclosure also provides methods for diagnosing and treating patientsbased on the presence or amount of PNH Type II cells in the patient. Forexample, the disclosure features a method for determining risk ofthrombocytopenia in a patient based on the percentage of PNH Type IIwhite blood cells detected in a biological sample from a patientsuspected of having PNH. The diagnostic methods described herein have anumber of advantages over prior art methods. For example, the methodsdescribed herein can more effectively separate PNH Type II white bloodcells from Type I cells, which allows for a more accurate and precisemeasurement of the percentage of the Type II cells in a biologicalsample, as well as a more accurate assessment of the total PNH clonesize, which comprises both Type II and Type III cells. In addition, PNHdiagnostic methods that rely on GPI-expression on Type II RBCs can beunreliable because of a high turnover of red blood cells (the inherentshorter life-span of PNH Type III RBCs due to elevated sensitivity tocomplement-mediated lysis) and frequent RBC transfusions received by PNHpatients. Therefore, the diagnostic methods described herein not onlyallow a practitioner to accurately and precisely quantify the percentageof Type II white blood cells in a biological sample, and thus the totalabnormal clone size in the sample, but the methods are also morereliable than prior methods that relied on detecting relatively unstablepopulations of PNH Type II RBCs.

In one aspect, the disclosure features a method for predicting whether apatient is at an increased risk for thrombosis. The method includesdetermining whether a patient is at an increased risk for thrombosisbased on the percentage of PNH Type II cells of the total number ofcells of the same histological type (same lineage) in a biologicalsample from the patient indicates that the patient is at an increasedrisk for thrombosis.

In another aspect, the disclosure features a method for predictingwhether a patient is likely to be thrombocytopenic. The method includesdetermining whether a patient is likely to be thrombocytopenic based onthe percentage of PNH Type II cells (e.g., Type II red blood cellsand/or Type II white blood cells) of the total number of cells of thesame histological type (same lineage) in a biological sample from thepatient indicates that the patient is likely to be thrombocytopenic.

In another aspect, the disclosure features a method for determiningwhether a patient is at an increased risk for thrombosis. The methodincludes providing (or receiving) information on the percentage of PNHType II cells of the total cells of the same histological type (samelineage) in a biological sample from a patient; and determining whethera patient is at an increased risk for thrombosis, wherein the percentageof PNH Type II cells of the total number of cells of the samehistological type in the biological sample indicates that the patient isat an increased risk for thrombosis.

In another aspect, the disclosure features a method for predictingwhether a patient is at risk for developing thrombosis, which methodincludes determining the percentage of PNH Type II cells in a biologicalsample from a patient; and providing a prediction of whether the patientis at an increased risk for thrombosis, wherein the percentage of PNHType II cells of the total number of cells of the same histological typein the biological sample indicates that the patient is at an increasedrisk for thrombosis.

In some embodiments, the PNH Type II cells are white blood cells (e.g.,granulocytes or monocytes). In some embodiments, the PNH Type II cellsare red blood cells.

In some embodiments of any of the methods described herein, thecombination of a percentage of PNH type II white blood cells that isgreater than or equal to 1.2% and a percentage of PNH type II red bloodcells that is greater than or equal to 0.02% is predictive of whetherthe patient is at an increased risk of developing thrombosis or islikely to be thrombocytopenic.

In some embodiments of any of the methods described herein, the patientis at an increased risk of developing thrombosis (and/or likely to bethrombocytopenic) when the percentage of PNH Type II white blood cellsis at least 1.2 (e.g., at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 65.3, or 70 or more) %.In some embodiments of any of the methods described herein, the patientis at an increased risk of developing thrombosis (and/or likely to bethrombocytopenic) when the percentage of PNH Type II red blood cells isat least 0.02 (e.g., at least 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8.2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 71, 71.3, or 75 or more) %.

In some embodiments of any of the methods described herein, a PNH TypeII white blood cell population that is between 1.2% to 65%, inclusive of1.2% and 65%, indicates that the patient is at an increased risk forthrombosis (and/or likely to be thrombocytopenic). In some embodiments,a PNH Type II white blood cell population that is greater than or equalto 5% indicates that the patient is at an increased risk for thrombosis(and/or likely to be thrombocytopenic). In some embodiments, a PNH TypeII white blood cell population that is greater than or equal to 10%,20%, or even 50% indicates that the patient is at an increased risk forthrombosis (and/or likely to be thrombocytopenic).

In some embodiments, any of the methods described herein can furtherinclude obtaining the biological sample from the patient. The biologicalsample can be, e.g., a whole blood sample.

In another aspect, the disclosure features a method for predictingwhether a patient is at an increased risk for developing thrombosis(and/or likely to be thrombocytopenic). The method includes determiningthe percentage of Type II white blood cells of the total white bloodcells of the same histological type in a biological sample from apatient; and predicting whether the patient is at an increased risk fordeveloping thrombosis, wherein the patient is at an increased risk fordeveloping thrombosis (and/or likely to be thrombocytopenic) if thepercentage of Type II white blood cells is greater than or equal to 1.2(e.g., greater than or equal to 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 65.3, or 70 or more) %.

In another aspect, the disclosure features a method for predictingwhether a patient is at an increased risk for developing thrombosis(and/or likely to be thrombocytopenic). The method includes determiningthe percentage of Type II red blood cells of the total red blood cellsof the same histological type in a biological sample from a patient; andpredicting whether the patient is at an increased risk for developingthrombosis (and/or likely to be thrombocytopenic), wherein the patientis at an increased risk for developing thrombosis (and/or likely to bethrombocytopenic) if the percentage of Type II red blood cells isgreater than or equal to 0.02 (e.g., greater than or equal to 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 71.3, or 75 or more) %.

In yet another aspect, the disclosure features a method for selecting atherapy for a patient, which method includes selecting one or both of ananti-thrombotic therapy and an anti-thrombocytopenic therapy for apatient determined to have a PNH Type II white blood cell population ofgreater than or equal to 1.2 (e.g., greater than or equal to 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 65.3, or 70 or more) % and/or a PNH Type II red blood cellpopulation of greater than or equal to 0.02 (e.g., greater than or equalto 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 71.3, or 75or more) %.

In another aspect, the disclosure features a method for treating apatient. The method includes administering to a patient in need thereofone or both of an anti-thrombotic therapy and an anti-thrombocytopenictherapy if the patient is determined to have a PNH Type II white bloodcell population of greater than or equal to 1.2 (e.g., greater than orequal to 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 65.3, or 70 or more) % and/or a PNH Type II redblood cell population of greater than or equal to 0.02 (e.g., greaterthan or equal to 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,71, 71.3, or 75 or more) %.

In some embodiments of any of the methods described herein, theanti-thrombocytopenic therapy can be, e.g., platelet transfusion.

In yet another aspect, the disclosure features a computer-based methodfor determining whether a patient is at an increased risk for developingthrombosis, which method includes receiving data including a medicalprofile of a PNH patient, the profile comprising information on thepercentage of PNH Type II white blood cells of the total white bloodcells of the same histological type (same lineage) in a biologicalsample from the patient; and processing at least the portion of the datacontaining the information to determine whether the patient is at anincreased risk for developing thrombosis, wherein the patient is at anincreased risk for developing thrombosis if the percentage of Type IIwhite blood cells is greater than or equal to 1.2 (e.g., greater than orequal to 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 65.3, or 70 or more) %.

In yet another aspect, the disclosure features a computer-based methodfor determining whether a patient is at an increased risk for developingthrombosis, which method includes receiving data including a medicalprofile of a PNH patient, the profile comprising information on thepercentage of PNH Type II red blood cells of the total red blood cellsof the same histological type (same lineage) in a biological sample fromthe patient; and processing at least the portion of the data containingthe information to determine whether the patient is at an increased riskfor developing thrombosis, wherein the patient is at an increased riskfor developing thrombosis if the percentage of Type II red blood cellsis greater than or equal to 0.02 (e.g., greater than or equal to 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 71.3, or 75 or more) %.

In another aspect, the disclosure features a computer-based method fordetermining whether a patient is at an increased risk for developingthrombosis, which method includes providing information on thepercentage of PNH Type II white blood cells of the total white bloodcells of the same histological type (same lineage) in a biologicalsample from the patient; inputting the information into a computer; andcalculating a parameter indicating whether the patient is at anincreased risk for thrombosis using the computer and the inputinformation, wherein the patient is at an increased risk for developingthrombosis if the percentage of Type II white blood cells is greaterthan or equal to 1.2 (e.g., greater than or equal to 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,65.3, or 70 or more) %.

In another aspect, the disclosure features a computer-based method fordetermining whether a patient is at an increased risk for developingthrombosis, which method includes providing information on thepercentage of PNH Type II white blood cells of the total white bloodcells of the same histological type in a biological sample from thepatient; inputting the information into a computer; and calculating aparameter indicating whether the patient is at an increased risk forthrombosis using the computer and the input information, wherein thepatient is at an increased risk for developing thrombosis if thepercentage of Type II white blood cells is greater than or equal to 0.02(e.g., greater than or equal to 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 71, 71.3, or 75 or more) %.

In some embodiments, any of the computer-based methods described hereincan further include storing the parameter on a computer-readable mediumand/or outputting the parameter.

In some embodiments, any of the methods described herein can include thestep of monitoring the patient for the development of at least onesymptom of thrombosis if the patient is at an increased risk ofdeveloping thrombosis. In some embodiments, any of the methods describedherein can include selecting an anti-thrombotic therapy for the patientif the patient is at an increased risk of developing thrombosis. In someembodiments, any of the methods described herein can includeadministering to the patient an anti-thrombotic therapy if the patientis at an increased risk for developing thrombosis. The anti-thrombotictherapy can be, e.g., an anticoagulant or a thrombolytic agent. Theanticoagulant can be, e.g., coumadin, heparin, or derivatives thereof.The thrombolytic agent can be, e.g., a tissue plasminogen activator,streptokinase, or a urokinase-type plasminogen activator.

In some embodiments of any of the methods described herein, a non-lyticvariant form of aerolysin protein can be used to determine thepercentage of PNH Type II white blood cells in the biological sample.

In some embodiments, any of the methods described herein can includerecording the determined percentage of PNH Type II cells in thebiological sample. In some embodiments, any of the methods describedherein can include recording the prediction of whether the patient is atan increased risk for developing thrombosis or whether the patient isnot at an increased risk for developing thrombosis. The recordation canbe on a computer-readable medium. The recordation can also be, e.g., ona tangible medium (e.g., a patient's physical record or chart).

In yet another aspect, the disclosure features a method for classifyingwhite blood cells. The method contacting a plurality of white bloodcells with a reagent that binds to: (i) GPI or (ii) a GPI-anchoredprotein; and classifying one or more of the white blood cells as PNHType II cells based on the amount of reagent bound to the cells.

In another aspect, the disclosure features a method for classifyingwhite blood cells, which method includes contacting a plurality of whiteblood cells with a reagent that binds to: (i) GPI or (ii) a GPI-anchoredprotein; interrogating at least a portion of the white blood cellscontacted with the reagent based on the amount of reagent bound to thecells; and classifying one or more of the interrogated cells as PNH TypeII cells.

In another aspect, the disclosure features a method for distinguishingbetween different white blood cell populations. The method includescontacting a plurality of white blood cells with a reagent that bindsto: (i) GPI or (ii) a GPI-anchored protein; and distinguishing at leasta portion of the white blood cells from other white blood cells of theplurality based on the amount of reagent bound the cells, wherein thePNH Type II white blood cells, if present, are sufficientlydistinguished from the Type I white blood cells and PNH Type III cellsof the same histological type (same lineage) to allow the percentage ofPNH Type II white blood cells of the total white blood cells of the samehistological type in the plurality to be determined. The method can alsoinclude determining the percentage of PNH Type II white blood cells.

In yet another aspect, the disclosure features a method for determiningthe percentage of PNH Type II white blood cells in a sample, whichmethod includes interrogating a plurality of white blood cells contactedwith a reagent based on the amount of reagent bound to the cells,wherein the reagent binds to: (i) GPI or (ii) a GPI-anchored protein,wherein the interrogating sufficiently distinguishes the PNH Type IIwhite blood cells, if present, from the Type I white blood cells and PNHType III cells of the same histological type to allow the percentage ofPNH Type II white blood cells of the total white blood cells of the samehistological type in the plurality to be determined; and determining thepercentage of PNH Type II white blood cells.

In some embodiments of any of the methods described herein, theplurality of white blood cells are contacted with a reagent that bindsto GPI and a reagent that binds to a GPI-linked protein.

In some embodiments of any of the methods described herein, thedistinguishing or interrogating of white blood cells (and/or thedetermination of the percentage of PNH Type II white blood cells)includes flow cytometry.

In some embodiments of any of the methods described herein, theplurality of white blood cells to be interrogated are obtained from apatient having, suspected of having, or at risk of developing PNH. Insome embodiments, the patient is one for whom a percentage of PNH TypeII red blood cells has been previously determined, but was suspectand/or inconclusive.

In some embodiments, any of the methods described herein can furtherinclude recording the percentage of PNH Type II white blood cells. Therecordation can be on a computer-readable medium or a tangible medium(e.g., a patient chart or record).

In some embodiments of any of the methods described herein, the reagentcan bind to a human GPI moiety. The reagent can be, e.g., an antibody oran antigen-binding fragment thereof, or an aerolysin protein. Theaerolysin protein can be, e.g., a variant form of aerolysin protein thatis non-lytic or is substantially non-lytic as compared to the wildtypeform of the protein. The non-lytic or substantially non-lytic aerolysinprotein can comprise the amino acid sequence depicted in SEQ ID NO:2 or7 wherein the threonine at position 253 is substituted with a cysteineand the alanine at position 300 is substituted for a cysteine.

In some embodiments of any of the methods described herein, the reagentcan bind to a GPI-anchored protein. For example, the reagent can be,e.g., an antibody or an antigen-binding fragment thereof that binds to aGPI-anchored protein. The GPI-anchored protein can be, e.g., alkalinephosphatase, 5′ nucleotidease acetylcholinesterase, dipeptidase, LFA-3,NCAM, PH-20, CD55, CD59, Thy-1, Qa-2, CD14, CD33, CD16 (the Fc_(γ)receptor III), carcinoembryonic antigen (CEA), CD24, CD66b, CD87, CD48,CD52, or any other GPI-anchored protein that is known in the art and/orset forth herein.

In some embodiments, a patient determined to have a PNH Type II whiteblood cell population of greater than or equal to 1.2% or a PNH Type IIred blood cell population of greater than or equal to 0.02% can bediagnosed as having PNH. In some embodiments, a patient diagnosed ashaving PNH or a previously diagnosed PNH patient who is determined tohave a PNH Type II white blood cell population greater than or equal to1.2% or a PNH Type II red blood cell population that is greater than orequal to 0.02% can be prescribed and/or treated with a complementinhibitor such as, but not limited to, eculizumab.

In yet another aspect, the disclosure features an antibody or anantigen-binding fragment thereof that binds to a human GPI moiety. Theantibody or antigen-binding fragment thereof can be, e.g., a recombinantantibody, a diabody, a chimerized or chimeric antibody, a deimmunizedhuman antibody, a fully human antibody, a single chain antibody, an Fvfragment, an Fd fragment, an Fab fragment, an Fab′ fragment, and anF(ab′)₂ fragment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the presently disclosed methods and compositions.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor determining risk of thrombocytopenia or thrombosis in a subject,will be apparent from the following description, the examples, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a two color dot plot depicting a population of humanperipheral blood granulocytes that were incubated with a solutioncontaining both a non-lytic variant of aerolysin conjugated with AlexaFluor® 488 and an antibody that binds to CD24 conjugated tophyocoerythrin (PE). The non-lytic aerolysin binds specifically to theGPI anchor, and therefore cells expressing any GPI-anchored proteins arelabeled with this fluorescent protein. CD24 is a GPI-linked proteinexpressed on granulocytes, so cells expressing CD24 will be bound byboth the anti-CD24 antibody and the non-lytic aerolysin. The X-axisrepresents the log intensity of detectable signal produced from theaerolysin conjugate bound to the cells and the Y-axis represents the logintensity of the detectable signal produced from the anti-CD24 antibodyconjugate bound to the cells. Three populations of granulocytes arerevealed by this analysis: Type III cells, which are devoid ofGPI-linked proteins and thus appear unlabeled with either the anti-CD24antibody and the non-lytic aerolysin; Type I granulocytes, which expresshigh levels of GPI-linked proteins relative to cells lackingGPI-anchors; and Type II granulocytes, which express intermediate levelsof GPI-linked proteins and thus are labeled with both the anti-CD24 andnon-lytic aerolysin at lower levels than those seen on normal (Type I)granulocytes.

FIG. 2 is a scatter plot depicting the absolute platelet count versusthe percentage of PNH Type II granulocytes in the blood of patients withPNH. The Y-axis represents the platelet count in 1 μL of patient blood(×10⁻³) and the X-axis represents the percentage of PNH Type IIgranulocytes within the total granulocyte population. The left half ofthe plot is a distribution of the platelet counts observed among PNHpatients (N=141) that have no detectable PNH Type II granulocytepopulations. The right half of the plot is a distribution of theplatelet counts observed among PNH patients (N=19) who have detectablePNH Type II granulocyte populations.

DETAILED DESCRIPTION

The present disclosure features a variety of diagnostic and therapeuticapplications that are useful for, inter alia, determining whether apatient has a PNH Type II cell population and/or is at an increased riskfor developing thrombocytopenia and/or thrombosis. The disclosure alsofeatures reagents that can be used in the methods. While in no wayintended to be limiting, exemplary reagents, conjugates, and methods forusing any of the foregoing are elaborated on below and are exemplifiedin the working Examples.

Reagents

The disclosure features a number of reagents that are useful in thediagnostic and therapeutic methods described herein. In someembodiments, the reagent binds to a glycosylphosphatidylinositol (GPI)moiety, which anchors many cell surface proteins to the cell membrane.GPI moieties generally contain a core ofethanolamine-HPO₄-6Manα1-2Manα1-6Manα1-4GlcNH₂₁-6myo-inositol-1HPO₄-diacyl-glycerol(or alkylacylglycerol or ceramide). See, e.g., Paulick and Bertozzi(2008) Biochemistry 47(27):6991-7000. However, a number of variations onthis core structure have been reported. For example, the glycan core canbe modified with side chains such as, but not limited to,phosphoethanolamine, mannose, galactose, sialic acid, or other sugars.Id.

In some embodiments, the reagent can be an aerolysin protein, e.g., anon-lytic aerolysin protein. Aerolysin is a channel-forming cytolyticprotein that is expressed by virulent Aeromonas species such as, but notlimited to, Aeromonas hydrophila and Aeromonas salmonicida. Aerolysin issecreted from the bacterial cell as a 52 kDa precursor that is convertedto the active form (activated) by proteolytic removal of a C-terminalpeptide. The aerolysin precursor can be activated by host proteases aswell as proteases secreted by an aerolysin-expressing bacterium. Oncebound to a cell, aerolysin oligomerizes to produce channels in, andultimately lyse, the cell (Howard and Buckley (1985) J Bacteriol163:336-340).

The amino acid sequences of the aerolysin polypeptide produced by eachof various members of the Aeromonas family are highly conserved.Accordingly, an aerolysin polypeptide, as used herein, can be from anyspecies of Aeromonas such as, but not limited to, A. hydrophila, A.caviae, A. veronii (biotype sobria), A. veronii (biotype veronii), A.jandaei, A. salmonicida, and A. schubertii.

In some embodiments, the aerolysin polypeptide is from A. hydrophila orA. salmonicida. In some embodiments, the aerolysin polypeptide is aproform containing a 24 amino acid signal peptide. In some embodiments,the proform aerolysin polypeptide can have, or consist of, a polypeptidehaving the amino acid sequence depicted in SEQ ID NO:1 or SEQ ID NO:6.

In some embodiments, the aerolysin polypeptide is a form of the proteinin which the signal sequence has been removed. For example, theaerolysin polypeptide can have, or consist of, a polypeptide having anamino acid sequence depicted in SEQ ID NO:2 or SEQ ID NO:7.

In some embodiments, the aerolysin polypeptide is an active form of theprotein. For example, the aerolysin polypeptide can have, or consist of,a polypeptide having an amino acid sequence depicted in SEQ ID NO:3.

As used herein, “polypeptide,” “peptide,” and “protein” are usedinterchangeably and mean any peptide-linked chain of amino acids,regardless of length or post-translational modification. The aerolysinpolypeptides described herein can contain or be wildtype proteins or canbe variants of the wild-type polypeptides that have not more than 50(e.g., not more than one, two, three, four, five, six, seven, eight,nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) conservative amino acidsubstitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine.

The aerolysin polypeptides described herein also include “GPI-bindingfragments” of the polypeptides, which are shorter than the full-length,proform polypeptides, but retain at least 10% (e.g., at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 50%, at least 55%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%, atleast 99.5%, or 100% or more) of the ability of the active polypeptideto bind to a GPI moiety. GPI-binding fragments of an aerolysinpolypeptide include terminal as well internal deletion variants of theprotein. Deletion variants can lack one, two, three, four, five, six,seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacid segments (of two or more amino acids) or non-contiguous singleamino acids. GPI-binding fragments can be at least 40 (e.g., at least41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, or325 or more) amino acid residues in length (e.g., at least 40 contiguousamino acid residues of SEQ ID Nos:1-3). In some embodiments, theGPI-binding fragment of an aerolysin polypeptide is less than 400 (e.g.,less than 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150,140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 60, 50, 49, 48, 47, 46, 45,44, 43, 42, 41, or 40) amino acid residues in length (e.g., less than400 contiguous amino acid residues of SEQ ID NOs:1-3). In someembodiments, the GPI-binding fragment of an aerolysin polypeptide is atleast 40, but less than 400, amino acid residues in length.

In some embodiments, the GPI-binding fragment of an aerolysinpolypeptide can include, or consist of, a polypeptide having thefollowing amino acid sequence: L D P D S F K H G D V T Q S D R Q L V K TV V G W A V N D S D T P Q S G Y D V T L R Y D T A T N W S K T N T Y G LS E K V T T K N K F K W P L V G E T E L S I E I A A N Q S W A S Q N G GS T T T S L S Q S V R P T V P A R S K I P V K I E L Y K A D I S Y P Y(SEQ ID NO:4).

In some embodiments, the GPI-binding fragment of an aerolysinpolypeptide can include, or consist of, a polypeptide having thefollowing amino acid sequence: L D P D S F K H G D V T Q S D R Q L V K TV V G W A V N D S D T P Q S G Y D V T L R Y D T A T N W S K T N T Y G LS E K V T T K N K F K W P L V G E C E L S I E I A A N Q S W A S Q N G GS T T T S L S Q S V R P T V P A R S K I P V K I E L Y K C D I S Y P Y(SEQ ID NO:5).

In some embodiments, the aerolysin polypeptide can have an amino acidsequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100) % identical to the aerolysin sequence havingthe amino acid sequence depicted in any one of SEQ ID NOs:1-3, 6, or 7(see below).

Percent (%) amino acid sequence identity is defined as the percentage ofamino acids in a candidate sequence that are identical to the aminoacids in a reference sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared can be determined by known methods.

Depending on the intended application, in some embodiments it may bepreferable to use a variant aerolysin polypeptide that lacks the abilityto lyse cells. Such variant forms of the aerolysin polypeptide are knownin the art and described in, e.g., Brodsky et al. (2000) Am J ClinPathol 114:459-466. In some embodiments, the non-lytic, variant form ofaerolysin contains, or consists of, the amino acid sequence depicted inSEQ ID NO:2 or SEQ ID NO:7 wherein one or more of: the histidine atposition 132 is substituted for an asparagine (His132Asn); the glycineat position 202 is a cysteine; the threonine at position 253 is acysteine and the alanine at position 300 is a cysteine; and thethreonine at position 225 is a glycine. One exemplary non-lytic variantof aerolysin comprises the amino acid sequence depicted in SEQ ID NOs: 2or 7, wherein the threonine at position 253 is a cysteine and thealanine at position 300 is a cysteine. As described above, the variantforms will retain the ability to bind to GPI moieties.

In some embodiments, the variant aerolysin polypeptide has less than 10(e.g., less than 9, 8, 7, 6, 5, 4, 3, 2, or 1) % of the ability of thenon-variant counterpart aerolysin polypeptide to lyse target cells. Insome embodiments, the variant aerolysin polypeptide has no detectablecytolytic activity.

Methods for determining whether a variant aerolysin polypeptide binds toa GPI moiety are known in the art and exemplified in the workingexamples. For example, cell-based methods for detecting the bindingbetween a variant aerolysin polypeptide and a GPI moiety on a cellsurface can be determined using flow cytometry techniques and adectectably-labeled (e.g., a fluorophore-labeled) variant aerolysinpolypeptide. See, e.g., Hong et al. (2002) EMBO J 21(19):5047-5056.

Likewise, methods for detecting and/or quantitating the cytolyticactivity of an aerolysin polypeptide or variant thereof are also knownin the art. For example, the hemolytic activity of a variant Aerolysinpolypeptide can be determined by contacting the variant polypeptide tonormal human erythrocytes and measuring the amount of hemoglobinreleased from the erythrocytes. See, e.g., Howard and Buckley (1982)Biochemistry 21(7):1662-1667; Avigad and Bernheimer (1976) Infection andImmunity 13(5):1378-1381; Garland and Buckley (1988) Infection andImmunity 56(5):1249-1253; and Bernheimer and Avigard (1974) Infectionand Immunity 9:1016-1021. A decreased amount, or the absence of,cytolytic activity by the variant, as compared to the amount ofcytolytic activity possessed by the non-variant counterpart polypeptide,is an indication that the variant polypeptide has reduced or absentcytolytic activity.

Methods for obtaining an aerolysin polypeptide, or producing a variantof the polypeptide as described herein, are known in the art ofmolecular biology and exemplified in the working Examples. See, e.g.,Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual, 2^(nd)Edition,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al. (1992) “Current Protocols in Molecular Biology,”Greene Publishing Associates. Template DNA encoding an aerolysinpolypeptide can be obtained from any of the Aeromonas species describedherein using standard techniques (see, e.g., Sambrook et al. (1989),supra). For example, Howard et al. describes the isolation andcharacterization of a nucleic acid sequence encoding an aerolysinpolypeptide from Aeromonas hydrophile (Howard et al. (1987) J Bacteriol169(6):2869-2871). An aerolysin polypeptide isolated from Aeromonassalmonicida is described in Buckley (1990) Biochem. Cell Biol.68:221-224 and Wong et al. (1989) J Bacteriol. 171:2523-2527.

In some embodiments, an aerolysin polypeptide can contain internal orterminal (carboxy or amino-terminal) irrelevant or heterologous aminoacid sequences (e.g., sequences derived from other proteins or syntheticsequences not corresponding to any naturally occurring protein). Thesequences can be, for example, an antigenic tag (e.g., FLAG,polyhistidine, hemagglutinin (HA), glutathione-S-transferase (GST), ormaltose-binding protein (MBP)). Heterologous sequences can also includeproteins useful as diagnostic or detectable markers, for example,luciferase, green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT).

Exemplary aerolysin polypeptides as well as methods for preparing andpurifying the polypeptides are described in U.S. provisional patentapplication Ser. No. 61/200,655, the disclosure of which is incorporatedherein by reference in its entirety.

In some embodiments, the reagent can be an antibody that binds to a GPImoiety. Antibodies that bind to non-human GPI moieties have beenidentified and isolated. See, e.g., Naik et al. (2006) Infection andImmunity 74(2):1412 (isolation of a naturally-occurring antibody thatbinds to the GPI moieties of Plasmodium falciparum.). As described indetail herein, it is well within the capability of an ordinarily skilledartisan to generate an antibody that binds to a human GPI moiety.

As used herein, the term “antibody” refers to a whole or intact antibodymolecule (e.g., IgM, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA,IgD, or IgE) or any antigen-binding fragment thereof. The term antibodyincludes, e.g., a chimerized or chimeric antibody, a humanized antibody,a deimmunized antibody, and a fully human antibody. Antigen-bindingfragments of an antibody include, e.g., a single chain antibody, asingle chain Fv fragment (scFv), an Fd fragment, an Fab fragment, anFab′ fragment, or an F(ab′)₂ fragment. An scFv fragment is a singlepolypeptide chain that includes both the heavy and light chain variableregions of the antibody from which the scFv is derived. In addition,intrabodies, minibodies, triabodies, and diabodies (see, e.g.,Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson andKortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994) Structure2(12):1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology51:257-283, the disclosures of each of which are incorporated herein byreference in their entirety) are also included in the definition ofantibody and are compatible for use in the methods described herein.Bispecific antibodies are also embraced by the term “antibody.”Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. Methods for generating an antibody or a fragment thereof arediscussed herein.

In some embodiments, the reagent can bind to a GPI-anchored protein. Forexample, the reagent can be an antibody that binds to a GPI-anchoredprotein. In some embodiments, the reagent can be a ligand for aGPI-anchored protein. GPI-anchored proteins are myriad and include,without limitation, alkaline phosphatase, 5′ nucleotideaseacetylcholinesterase, dipeptidase, LFA-3, NCAM, PH-20, CD55, CD59,Thy-1, Qa-2, CD14, CD33, CD16 (the Fc_(γ) receptor III),carcinoembryonic antigen (CEA), and CD52. Antibodies that bind toGPI-anchored proteins are well known in the art and are described in,e.g., Hall and Rosse (1996) supra, Richards et al. (2008) Cytometry BClin Cytom 76B(1):47-55; Richards and Barnett (2007) Clin Lab Med27(3):577-590; Luzzatto et al. (2006) Int J Hematol 84(2):104-112; andThomason et al. (2004) Am J Clin Pathol 122(1):128-134. Such antibodiesare also commercially available from, e.g., Santa Cruz Biotechology,Inc. (Santa Cruz, Calif.), Novus Biologicals (Littleton, Colo.), and R&D Systems (Minneapolis, Minn.).

Suitable methods for generating an antibody that binds to a GPI-anchoredprotein or a GPI moiety for use in the diagnostic and/or therapeuticmethods described are well known in the art and described in thefollowing section.

Methods for Generating an Antibody

Suitable methods for producing an antibody (e.g., an antibody that bindsto a GPI moiety or a GPI-anchored protein), or antigen-binding fragmentsthereof, in accordance with the disclosure are known in the art anddescribed herein. For example, monoclonal anti-CD55 antibodies may begenerated using human CD55-expressing cells, a CD55 polypeptide, or anantigenic fragment of CD55 polypeptide, as an immunogen, thus raising animmune response in animals from which antibody-producing cells and inturn monoclonal antibodies may be isolated. The sequence of suchantibodies may be determined and the antibodies or variants thereofproduced by recombinant techniques. Recombinant techniques may be usedto produce chimeric, CDR-grafted, humanized and fully human antibodiesbased on the sequence of the monoclonal antibodies as well aspolypeptides capable of binding to a GPI-anchored protein or a GPImoiety.

Moreover, antibodies derived from recombinant libraries (“phageantibodies”) may be selected using, e.g., cells expressing a GPI moietyor a GPI-anchored protein, recombinant GPI-linked proteins, or free GPImoieties as bait to isolate the antibodies or polypeptides on the basisof target specificity. The production and isolation of non-human andchimeric antibodies are well within the purview of the skilled artisan.

Recombinant DNA technology can be used to modify one or morecharacteristics of the antibodies produced in non-human cells. Thus,chimeric antibodies can be constructed in order to decrease theimmunogenicity thereof in diagnostic or therapeutic applications.Moreover, immunogenicity can be minimized by humanizing the antibodiesby CDR grafting and, optionally, framework modification. See, U.S. Pat.Nos. 5,225,539 and 7,393,648, the contents of each of which areincorporated herein by reference.

Antibodies can be obtained from animal serum or, in the case ofmonoclonal antibodies or fragments thereof, produced in cell culture.Recombinant DNA technology can be used to produce the antibodiesaccording to established procedure, including procedures in bacterial orpreferably mammalian cell culture. The selected cell culture systempreferably secretes the antibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g. E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to polycistronic (e.g., bicistronic) DNA sequences encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 xYT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up production to give large amounts of the desiredantibodies. Techniques for bacterial cell, yeast, plant, or mammaliancell cultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibers,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules are described in the above references and also in, e.g.:WO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith(1985) Science 225:1315-1317; Parmley and Smith (1988) Gene 73:305-318;De La Cruz et al. (1988) Journal of Biological Chemistry 263:4318-4322;U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409; WO88/06630;WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No. 5,571,698; U.S. Pat.No. 6,040,136; Davis et al. (1999) Cancer Metastasis Rev. 18(4):421-5;and Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295; Tomizukaet al. (2000) Proc. Natl. Acad. Sci. USA 97(2): 722-727, the contents ofeach of which are incorporated herein by reference in their entirety.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of GPI or GPI-anchoredprotein-expressing cells, by immunoblotting, by an enzyme immunoassay,e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g. byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with a GPI-moiety, a cell expressing GPI-anchors at itssurface, a GPI-anchored polypeptide, a cell expressing a GPI-anchoredprotein at its surface, or with Protein-A or -G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against a GPI moiety or aGPI-anchored protein in a suitable mammal. For example, a rabbit isimmunized with a GPI-moiety, a cell expressing GPI-anchors at itssurface, a GPI-anchored polypeptide, a cell expressing a GPI-anchoredprotein at its surface, or fragments thereof. A phage display libraryproduced from the immunized rabbit is constructed and panned for thedesired antibodies in accordance with methods well known in the art(such as, e.g., the methods disclosed in the various referencesincorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are also disclosed.The preferred hybridoma cells are genetically stable, secrete monoclonalantibodies described herein of the desired specificity, and can beexpanded from deep-frozen cultures by thawing and propagation in vitroor as ascites in vivo.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies against a GPI moietyor a GPI-anchored protein. In that process, a suitable mammal, forexample a Balb/c mouse, is immunized with one or more polypeptides orantigenic fragments of, e.g., CD55 or CD14 or with one or morepolypeptides or antigenic fragments derived from a CD55-expressing cell,the CD55-expressing cell itself, or an antigenic carrier containing apurified polypeptide as described. Similarly, the mammal can beimmunized with a human GPI moiety, a fragment thereof, or cells thatexpress the human GPI moiety, perhaps at a high amount.Antibody-producing cells of the immunized mammal are grown briefly inculture or fused with cells of a suitable myeloma cell line. The hybridcells obtained in the fusion are cloned, and cell clones secreting thedesired antibodies are selected. For example, spleen cells of Balb/cmice immunized with, e.g., a GPI-anchored protein or a GPI moiety arefused with cells of the myeloma cell line PAI or the myeloma cell lineSp2/0-Ag 14. The obtained hybrid cells are then screened for secretionof the desired antibodies and positive hybridoma cells are cloned.

Methods for preparing a hybridoma cell line include immunizing Balb/cmice by injecting subcutaneously and/or intraperitoneally an antigen ofinterest several times, e.g., four to six times, over several months,e.g., between two and four months. Spleen cells from the immunized miceare taken two to four days after the last injection and fused with cellsof the myeloma cell line PAI in the presence of a fusion promoter,preferably polyethylene glycol. Preferably, the myeloma cells are fusedwith a three- to twenty-fold excess of spleen cells from the immunizedmice in a solution containing about 30% to about 50% polyethylene glycolof a molecular weight around 4000. After the fusion, the cells areexpanded in suitable culture media as described supra, supplemented witha selection medium, for example HAT medium, at regular intervals inorder to prevent normal myeloma cells from overgrowing the desiredhybridoma cells.

The antibodies and fragments thereof can be “chimeric.” Chimericantibodies and antigen-binding fragments thereof comprise portions fromtwo or more different species (e.g., mouse and human). Chimericantibodies can be produced with mouse variable regions of desiredspecificity spliced into human constant domain gene segments (forexample, U.S. Pat. No. 4,816,567). In this manner, non-human antibodiescan be modified to make them more suitable for human clinicalapplication (e.g., methods for detecting Type II PNH cells).

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (e.g., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a non-human source. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Methods ofpreparing humanized antibodies are generally well known in the art. Forexample, humanization can be essentially performed following the methodof Winter and co-workers (see, e.g., Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyenet al. (1988) Science 239:1534-1536), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Also see,e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In someembodiments, humanized forms of non-human (e.g., mouse) antibodies arehuman antibodies (recipient antibody) in which hypervariable (CDR)region residues of the recipient antibody are replaced by hypervariableregion residues from a non-human species (donor antibody) such as amouse, rat, rabbit, or non-human primate having the desired specificity,affinity, and binding capacity. In some instances, framework regionresidues of the human immunoglobulin are also replaced by correspondingnon-human residues (so called “back mutations”). In addition, phagedisplay libraries can be used to vary amino acids at chosen positionswithin the antibody sequence. The properties of a humanized antibody arealso affected by the choice of the human framework. Furthermore,humanized and chimerized antibodies can be modified to comprise residuesthat are not found in the recipient antibody or in the donor antibody inorder to further improve antibody properties, such as, for example,affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human germline immunoglobulinsequences. Human antibodies can include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences (i.e., humanized antibodies). Fully human or human antibodiesmay be derived from transgenic mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), and constant (C)exons) or from human cells. For example, it is now possible to producetransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. (See, e.g., Jakobovits et al.(1993) Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al. (1993)Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; andDuchosal et al. (1992) Nature 355:258.) Transgenic mice strains can beengineered to contain gene sequences from unrearranged humanimmunoglobulin genes. The human sequences may code for both the heavyand light chains of human antibodies and would function correctly in themice, undergoing rearrangement to provide a wide antibody repertoiresimilar to that in humans. The transgenic mice can be immunized with thetarget protein (e.g., a GPI moiety or a GPI-anchored protein such asCD55 or CD14) to create a diverse array of specific antibodies and theirencoding RNA. Nucleic acids encoding the antibody chain components ofsuch antibodies may then be cloned from the animal into a displayvector. Typically, separate populations of nucleic acids encoding heavyand light chain sequences are cloned, and the separate populations thenrecombined on insertion into the vector, such that any given copy of thevector receives a random combination of a heavy and a light chain. Thevector is designed to express antibody chains so that they can beassembled and displayed on the outer surface of a display packagecontaining the vector. For example, antibody chains can be expressed asfusion proteins with a phage coat protein from the outer surface of thephage. Thereafter, display packages can be screened for display ofantibodies binding to a target.

In addition, human antibodies can be derived from phage-displaylibraries (Hoogenboom et al. (1991) J. Mol. Biol. 227:381; Marks et al.(1991) J. Mol. Biol., 222:581-597; and Vaughan et al. (1996) NatureBiotech 14:309 (1996)). Synthetic phage libraries can be created whichuse randomized combinations of synthetic human antibody V-regions. Byselection on antigen fully human antibodies can be made in which theV-regions are very human-like in nature. See, e.g., U.S. Pat. Nos.6,794,132, 6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma2:361-367, the contents of each of which are incorporated herein byreference in their entirety.

For the generation of human antibodies, also see Mendez et al. (1998)Nature Genetics 15:146-156, Green and Jakobovits (1998) J. Exp. Med.188:483-495, the disclosures of which are hereby incorporated byreference in their entirety. Human antibodies are further discussed anddelineated in U.S. Pat. Nos. 5,939,598; 6,673,986; 6,114,598; 6,075,181;6,162,963; 6,150,584; 6,713,610; and 6,657,103 as well as U.S. PatentPublication Nos. 20030229905 A1, 20040010810 A1, US 20040093622 A1,20060040363 A1, 20050054055 A1, 20050076395 A1, 20050287630 A1. See alsoInternational Publication Nos. WO 94/02602, WO 96/34096, and WO98/24893, and European Patent No. EP 0 463 151 B1. The disclosures ofeach of the above-cited patents, applications, and references are herebyincorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,825;5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; and 5,814,318;5,591,669; 5,612,205; 5,721,367; 5,789,215; 5,643,763; 5,569,825;5,877,397; 6,300,129; 5,874,299; 6,255,458; and 7,041,871, thedisclosures of which are hereby incorporated by reference. See alsoEuropean Patent No. 0 546 073 B1, International Patent Publication Nos.WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, thedisclosures of each of which are hereby incorporated by reference intheir entirety. See further Taylor et al. (1992) Nucleic Acids Res. 20:6287; Chen et al. (1993) Int. Immunol. 5: 647; Tuaillon et al. (1993)Proc. Natl. Acad. Sci. USA 90: 3720-4; Choi et al. (1993) NatureGenetics 4: 117; Lonberg et al. (1994) Nature 368: 856-859; Taylor etal. (1994) International Immunology 6: 579-591; Tuaillon et al. (1995)J. Immunol. 154: 6453-65; Fishwild et al. (1996) Nature Biotechnology14: 845; and Tuaillon et al. (2000) Eur. J. Immunol. 10: 2998-3005, thedisclosures of each of which are hereby incorporated by reference intheir entirety.

In certain embodiments, de-immunized antibodies (e.g., antibodies thatbind to a human GPI moiety or to a GPI-anchored protein) orantigen-binding fragments thereof are provided. De-immunized antibodiesor antigen-binding fragments thereof may be modified so as to render theantibody or antigen-binding fragment thereof non-immunogenic, or lessimmunogenic, to a given species. De-immunization can be achieved bymodifying the antibody or antigen-binding fragment thereof utilizing anyof a variety of techniques known to those skilled in the art (see, e.g.,PCT Publication Nos. WO 04/108158 and WO 00/34317). For example, anantibody or antigen-binding fragment thereof may be de-immunized byidentifying potential T cell epitopes and/or B cell epitopes within theamino acid sequence of the antibody or antigen-binding fragment thereofand removing one or more of the potential T cell epitopes and/or B cellepitopes from the antibody or antigen-binding fragment thereof, forexample, using recombinant techniques. The modified antibody orantigen-binding fragment thereof may then optionally be produced andtested to identify antibodies or antigen-binding fragments thereof thathave retained one or more desired biological activities, such as, forexample, binding affinity, but have reduced immunogenicity. Methods foridentifying potential T cell epitopes and/or B cell epitopes may becarried out using techniques known in the art, such as, for example,computational methods (see e.g., PCT Publication No. WO 02/069232), invitro or in silico techniques, and biological assays or physical methods(such as, for example, determination of the binding of peptides to MHCmolecules, determination of the binding of peptide:MHC complexes to theT cell receptors from the species to receive the antibody orantigen-binding fragment thereof, testing of the protein or peptideparts thereof using transgenic animals with the MHC molecules of thespecies to receive the antibody or antigen-binding fragment thereof, ortesting with transgenic animals reconstituted with immune system cellsfrom the species to receive the antibody or antigen-binding fragmentthereof, etc.). In various embodiments, the de-immunized antibodiesdescribed herein include de-immunized antigen-binding fragments, Fab,Fv, scFv, Fab′ and F(ab′)₂, monoclonal antibodies, murine antibodies,engineered antibodies (such as, for example, chimeric, single chain,CDR-grafted, humanized, fully human antibodies, and artificiallyselected antibodies), synthetic antibodies and semi-syntheticantibodies.

In some embodiments, a recombinant DNA comprising an insert coding for aheavy chain variable domain and/or for a light chain variable domain ofan antibody-expressing cell line is produced. The term DNA includescoding single stranded DNAs, double stranded DNAs consisting of saidcoding DNAs and of complementary DNAs thereto, or these complementary(single stranded) DNAs themselves.

Furthermore, a DNA encoding a heavy chain variable domain and/or a lightchain variable domain of an antibody (e.g., an anti-GPI antibody or ananti-GPI-anchored protein antibody) can be enzymatically or chemicallysynthesized to contain the authentic DNA sequence coding for a heavychain variable domain and/or for the light chain variable domain, or amutant thereof. A mutant of the authentic DNA is a DNA encoding a heavychain variable domain and/or a light chain variable domain of theabove-mentioned antibodies in which one or more amino acids are deleted,inserted, or exchanged with one or more other amino acids. Preferablysaid modification(s) are outside the CDRs of the heavy chain variabledomain and/or of the light chain variable domain of the antibody inhumanization and expression optimization applications. The term mutantDNA also embraces silent mutants wherein one or more nucleotides arereplaced by other nucleotides with the new codons coding for the sameamino acid(s). The term mutant sequence also includes a degeneratesequence. Degenerate sequences are degenerate within the meaning of thegenetic code in that an unlimited number of nucleotides are replaced byother nucleotides without resulting in a change of the amino acidsequence originally encoded. Such degenerate sequences may be useful dueto their different restriction sites and/or frequency of particularcodons which are preferred by the specific host, particularly E. coli,to obtain an optimal expression of the heavy chain murine variabledomain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an antibody of interest fused to a human constantdomain IgG, for example γ1, γ2, γ3 or γ4, in particular embodiments γ1or γ4, may be used. Recombinant DNAs including an insert coding for alight chain murine variable domain of an antibody fused to a humanconstant domain κ or λ, preferably κ, are also provided.

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA sequence encoding a peptide facilitatingthe purification of the antibody and/or a cleavage site and/or a peptidespacer and/or an agent. The DNA coding for an agent is intended to be aDNA coding for the agent useful in diagnostic or therapeuticapplications. Thus, agent molecules which are toxins or enzymes,especially enzymes capable of catalyzing the activation of prodrugs, areparticularly indicated. The DNA encoding such an agent has the sequenceof a naturally occurring enzyme or toxin encoding DNA, or a mutantthereof, and can be prepared by methods well known in the art.

Accordingly, the monoclonal antibodies or antigen-binding fragments ofthe disclosure can be naked antibodies or antigen-binding fragments thatare not conjugated to other agents, for example, a therapeutic agent ordetectable label. Alternatively, the monoclonal antibody orantigen-binding fragment can be conjugated to an agent such as, forexample, a cytotoxic agent, a small molecule, a hormone, an enzyme, agrowth factor, a cytokine, a ribozyme, a peptidomimetic, a chemical, aprodrug, a nucleic acid molecule including coding sequences (such asantisense, RNAi, gene-targeting constructs, etc.), or a detectable label(e.g., an NMR or X-ray contrasting agent, fluorescent molecule, etc.).In certain embodiments, an antibody or an antigen-binding fragmentthereof (e.g., Fab, Fv, single-chain scFv, Fab′, and F(ab′)₂) is linkedto a molecule that increases the half-life of the antibody orantigen-binding fragment (see the section entitled “Conjugates”).

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors relies upon the integration of the desired gene sequencesinto the host cell genome. Cells which have stably integrated DNA can beselected by simultaneously introducing drug resistance genes such as E.coli gpt (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA, 78:2072)or Tn5 neo (Southern and Berg (1982) Mol. Appl. Genet. 1:327). Theselectable marker gene can be either linked to the DNA gene sequences tobe expressed, or introduced into the same cell by co-transfection(Wigler et al. (1979) Cell 16:77). A second class of vectors utilizesDNA elements which confer autonomously replicating capabilities to anextrachromosomal plasmid. These vectors can be derived from animalviruses, such as bovine papillomavirus (Sarver et al. (1982) Proc. Natl.Acad. Sci. USA, 79:7147), polyoma virus (Deans et al. (1984) Proc. Natl.Acad. Sci. USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature293:79).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein, additional gene expressionelements regulating transcription of the gene and processing of the RNAare required for the synthesis of immunoglobulin mRNA. These elementsmay include splice signals, transcription promoters, including induciblepromoters, enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama and Berg(1983) Mol. Cell Biol. 3:280; Cepko et al. (1984) Cell 37:1053; andKaufman (1985) Proc. Natl. Acad. Sci. USA 82:689.

As is evident from the disclosure, the anti-GPI moiety antibodies oranti-GPI-anchored protein antibodies can be used in methods fordiagnosing disease (e.g., diagnosing PNH or an increased risk ofdeveloping thrombocytopenia), monitoring of disease progression, and theselection of appropriate therapies, including combination therapies, fortreating PNH, thrombocytopenia, or thrombosis in a subject.

In the diagnostic embodiments of the present disclosure, bispecificantibodies are contemplated. Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for a GPI moiety or a GPI-anchoredprotein on a cell (such as, e.g., a white blood cell or a red bloodcell), the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello (1983) Nature305:537-539). Antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, including at least partof the hinge, C_(H)2, and C_(H)3 regions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism. For further details ofillustrative currently known methods for generating bispecificantibodies see, e.g., Suresh et al. (1986) Methods in Enzymology121:210; PCT Publication No. WO 96/27011; Brennan et al. (1985) Science229:81; Shalaby et al., J. Exp. Med. (1992) 175:217-225; Kostelny et al.(1992) J. Immunol. 148(5):1547-1553; Hollinger et al. (1993) Proc. Natl.Acad. Sci. USA 90:6444-6448; Gruber et al. (1994) J. Immunol. 152:5368;and Tutt et al. (1991) J. Immunol. 147:60. Bispecific antibodies alsoinclude cross-linked or heteroconjugate antibodies. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. (See, e.g., Kostelny et al. (1992) J. Immunol.148(5):1547-1553). The leucine zipper peptides from the Fos and Junproteins may be linked to the Fab′ portions of two different antibodiesby gene fusion. The antibody homodimers may be reduced at the hingeregion to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. (See, e.g., Gruber et al. (1994) J. Immunol. 152:5368.)Alternatively, the antibodies can be “linear antibodies” as describedin, e.g., Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly,these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Conjugates

In some embodiments, a reagent described herein (e.g., a non-lyticaerolysin polypeptide or an antibody that binds to a GPI moiety or aGPI-anchored protein) can be conjugated to a heterologous moiety. Theheterologous moiety can be, e.g., a heterologous protein (see above), atherapeutic agent (e.g., a toxin or a drug), or a detectable label suchas, but not limited to, a radioactive label, an enzymatic label, afluorescent label, or a luminescent label. Suitable radioactive labelsinclude, e.g., ³²P, ³³P, ¹⁴C, ¹²⁵I, ¹³¹I, ³⁵S, and ³H. Suitablefluorescent labels include, without limitation, fluorescein, fluoresceinisothiocyanate (FITC), Alexa Fluor® 488, Alexa Fluor® 647, GFP, DyLight488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor®700, Cy5, allophycocyanin, Cy7, and PE-Alexa Fluor® 750. Luminescentlabels include, e.g., any of a variety of luminescent lanthanide (e.g.,europium or terbium) chelates. For example, suitable europium chelatesinclude the europium chelate of diethylene triamine pentaacetic acid(DTPA). Enzymatic labels include, e.g., alkaline phosphatase, CAT,luciferase, and horseradish peroxidase.

Suitable methods for conjugating a heterologous moiety to the reagentare well-known in the art of protein chemistry. For example, twoproteins can be cross-linked using any of a number of known chemicalcross linkers. Examples of such cross linkers are those which link twoamino acid residues via a linkage that includes a “hindered” disulfidebond. In these linkages, a disulfide bond within the cross-linking unitis protected (by hindering groups on either side of the disulfide bond)from reduction by the action, for example, of reduced glutathione or theenzyme disulfide reductase. One suitable reagent,4-succinimidyloxycarbonyl-α-methyl-α (2-pyridyldithio)toluene (SMPT),forms such a linkage between two proteins utilizing a terminal lysine onone of the proteins and a terminal cysteine on the other.Heterobifunctional reagents that cross-link by a different couplingmoiety on each protein can also be used. Other useful cross-linkersinclude, without limitation, reagents which link two amino groups (e.g.,N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g.,1,4-bis-maleimidobutane) an amino group and a sulfhydryl group (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and acarboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an aminogroup and a guanidinium group that is present in the side chain ofarginine (e.g., p-azidophenyl glyoxal monohydrate).

Radioactive labels can be conjugated to the reagent by covalent ornon-covalent (e.g., ionic or hydrophobic) bonds. They can be bound toany part of the protein provided that the conjugation does not interferewith the ability of the reagent to bind to a GPI moiety or to aGPI-anchored protein. In some embodiments, where the reagent is aprotein, the radioactive label can be directly conjugated to the aminoacid backbone of the reagent. Alternatively, the radioactive label canbe included as part of a larger molecule (e.g., ¹²⁵I inmeta-[¹²⁵I]iodophenyl-N-hydroxysuccinimide ([¹²⁵ I]mIPNHS) which bindsto free amino groups to form meta-iodophenyl (mIP) derivatives ofrelevant proteins (see, e.g., Rogers et al. (1997) J. Nucl. Med.38:1221-1229) or chelate (e.g., to DOTA or DTPA) which is in turn boundto the protein backbone. Methods of conjugating the radioactive labelsor larger molecules/chelates containing them to the reagents describedherein are also known in the art. Such methods involve incubating thereagent with the radioactive label under conditions (e.g., pH, saltconcentration, and/or temperature) that facilitate binding of theradioactive label or chelate to the reagent (see, e.g. U.S. Pat. No.6,001,329, the disclosure of which is incorporated herein by referencein its entirety).

Methods for conjugating a fluorescent label (sometimes referred to as a“fluorophore”) to a reagent (e.g., a non-lytic aerolysin protein or anantibody) are known in the art of protein chemistry. For example,fluorophores can be conjugated to free amino groups (e.g., of lysines)or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl(NHS) ester or TFP ester moieties attached to the fluorophores. In someembodiments, the fluorophores can be conjugated to a heterobifunctionalcross-linker moiety such as sulfo-SMCC. Suitable conjugation methodsinvolve incubating the reagent with the fluorophore under conditionsthat facilitate binding of the fluorophore to the reagent. See, e.g.,Welch and Redvanly (2003) “Handbook of Radiopharmaceuticals:Radiochemistry and Applications,” John Wiley and Sons (ISBN:0471495603). A variety of kits are commercially available for use inconjugating a fluorophore to a protein, e.g., the Alexa Fluor® 488Protein Labeling Kit and the Alexa Fluor® 647 Protein Labeling Kit(Molecular Probes, Invitrogen™) In some embodiments, the fluorophore canbe conjugated to a reagent at 1-2 mol dye per mol of protein.

In some embodiments, the reagents (e.g., an aerolysin protein or anantibody that binds to a GPI moiety or a GPI-anchored protein) can bemodified, e.g., with a moiety that improves the stabilization and/orretention of the antibodies themselves in circulation, e.g., in blood,serum, or other tissues. For example, a reagent described herein can bePEGylated as described in, e.g., Lee et al. (1999) Bioconjug. Chem10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews54:459-476. The stabilization moiety can improve the stability, orretention of, the reagent in a subject's body (e.g., blood or tissue) byat least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 ormore) fold.

Biological Samples and Sample Collection

Suitable biological samples for use in the methods described hereininclude any biological fluid, population of cells, or tissue or fractionthereof, which includes one or more white blood cells and/or one or morered blood cells. A biological sample can be, for example, a specimenobtained from a subject (e.g., a mammal such as a human) or can bederived from such a subject. For example, a sample can be a tissuesection obtained by biopsy, or cells that are placed in or adapted totissue culture. A biological sample can also be a biological fluid suchas urine, whole blood or a fraction thereof (e.g., plasma), saliva,semen, sputum, cerebral spinal fluid, tears, or mucus. A biologicalsample can be further fractionated, if desired, to a fraction containingparticular cell types. For example, a whole blood sample can befractionated into serum or into fractions containing particular types ofblood cells such as red blood cells or white blood cells (leukocytes).If desired, a biological sample can be a combination of differentbiological samples from a subject such as a combination of a tissue andfluid sample.

The biological samples can be obtained from a subject, e.g., a subjecthaving, suspected of having, or at risk of developing, paroxysmalnocturnal hemoglobinuria (PNH). Any suitable methods for obtaining thebiological samples can be employed, although exemplary methods include,e.g., phlebotomy, swab (e.g., buccal swab), lavage, or fine needleaspirate biopsy procedure. Non-limiting examples of tissues susceptibleto fine needle aspiration include lymph node, lung, thyroid, breast, andliver. Biological samples can also be obtained from bone marrow. Samplescan also be collected, e.g., by microdissection (e.g., laser capturemicrodissection (LCM) or laser microdissection (LMD)), bladder wash,smear (PAP smear), or ductal lavage.

Methods for obtaining and/or storing samples that preserve the activityor integrity of cells in the biological sample are well known to thoseskilled in the art. For example, a biological sample can be furthercontacted with one or more additional agents such as appropriate buffersand/or inhibitors, including protease inhibitors, the agents meant topreserve or minimize changes in the cells (e.g., changes in osmolarityor pH) or denaturation of cell surface proteins (e.g., GPI-linkedproteins) or GPI moieties on the surface of the cells. Such inhibitorsinclude, for example, chelators such as ethylenediamine tetraacetic acid(EDTA), ethylene glycol tetraacetic acid (EGTA), protease inhibitorssuch as phenylmethylsulfonyl fluoride (PMSF), aprotinin, and leupeptin.Appropriate buffers and conditions for storing or otherwise manipulatingwhole cells are described in, e.g., Pollard and Walker (1997), “BasicCell Culture Protocols,” volume 75 of Methods in Molecular Biology,Humana Press; Masters (2000) “Animal cell culture: a practicalapproach,” volume 232 of Practical approach series, Oxford UniversityPress; and Jones (1996) “Human cell culture protocols,” volume 2 ofMethods in molecular medicine, Humana Press.”

A sample also can be processed to eliminate or minimize the presence ofinterfering substances. For example, a biological sample can befractionated or purified to remove one or more materials (e.g., cells)that are not of interest. Methods of fractionating or purifying abiological sample include, but are not limited to, flow cytometry,fluorescence activated cell sorting, and sedimentation.

Diagnostic and Therapeutic Methods

As noted above and elaborated on in the working examples, the inventorshave discovered a clinical relationship between the presence or amountof PNH Type II hematopoietic cells (e.g., Type II white blood cellsand/or Type II red blood cells) and thrombocytopenia in a patient. Forexample, the inventors have determined that a patient with a PNH Type IIwhite blood cell population of at least 1.2 (e.g., at least 1.2, 1.5, 2,3, 5, 7, 9, 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45,47, 50, 52, 55, 57, 60, 62, 65, or 65.3 or more) % as compared to thetotal white blood cells of the same histological type (the same lineage)in the biological sample tested is much more likely to bethrombocytopenic than a patient who does not have a detectable PNH TypeII white blood cell population or a patient with a PNH Type II whiteblood cell population lower than 1.2%. Patient samples with PNH Type IIgranulocyte populations had similar peripheral white blood cell counts,peripheral red blood cell counts, absolute neutrophil counts, andhemoglobin (Hgb) levels, compared to patient samples without detectableType II granulocyte populations, indicating that differences in plateletcounts are likely not due to differences in underlying bone marrowproduction. In other words, the decreased platelet counts in patientswith detectable PNH Type II granulocyte clones may be due to increasedterminal complement-mediated platelet consumption or destruction, whichcan be associated with thrombosis, the leading cause of death among PNHpatients. See, e.g., Franchini (2006) Hematology 11(3):139-146.Accordingly, the present disclosure features methods for usinginformation related to the percentage of PNH Type II white blood cellsin a patient sample for determining whether the patient is at anincreased risk of developing thrombocytopenia and/or thrombosis.Similarly, the inventors have determined that a patient with a PNH TypeII RBC population of at least 0.02 (e.g., at least 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 71.3, or 75 or more) % is muchmore likely to be thrombocytopenic than a patient who does not have adetectable PNH Type II RBC population or a patient with a PNH Type IIRBC population lower than 0.02%. Thus, the present disclosure alsofeatures methods for using information related to the PNH Type II RBCclone size in a patient for determining whether the patient is at riskof developing thrombocytopenia and/or thrombosis.

The following methods can be employed to detect the presence, or todetermine the percentage, of PNH Type II hematopoietic cells (e.g., TypeII PNH white blood cells) as compared to the total amount of cells ofthe same histological type (same lineage) in a biological sample fromthe patient. In embodiments where a plurality of white blood cells areinterrogated, the methods are useful in allowing a practitioner todistinguish between populations of PNH Type I, Type II, and Type IIIwhite blood cells of the same histological type (same lineage) in orderto accurately determine the size of the total abnormal PNH population(i.e., Type II plus Type III cells) as compared to the total number ofwhite blood cells of the same histological type in the plurality (and/orallow the practitioner to determine the percentage of PNH Type II whiteblood cells in the plurality). First, a population of cells (e.g., whiteblood cells, red blood cells, or a combination of white and red bloodcells) is contacted with a reagent that binds to: (i) a GPI moiety or(ii) a GPI-linked protein for a period of time and under conditions thatallow for the binding of the reagent to the GPI moiety or GPI-linkedprotein if present on the surface of cells present in the sample. Thepopulation of cells can be present in a biological sample (e.g., a wholeblood sample; see the section entitled “Biological Samples and SampleCollection”), e.g., a biological sample obtained from a patient. Forexample, cells present in a whole blood sample from a patient can becontacted with an aerolysin protein (e.g., a non-lytic form ofaerolysin) or an antibody that binds to a GPI moiety or a GPI-anchoredprotein such as CD59. See, e.g., Hall and Rosse (1996) Blood87(12):5332-5340 and U.S. Pat. No. 6,593,095. At least a portion of thecells (e.g., white blood cells or RBCs) contacted with the reagent canbe distinguished based on the amount of reagent bound to the surface ofthe cells. For example, where a detectably-labeled reagent was used, theamount of reagent bound to the surface can be determined as a functionof the total amount of signal produced from detectably labeled reagentbound to the surface of the cell. As described above, the amount ofbinding of the reagent to the cell reflects the amount of expression ofGPI moieties and/or GPI-anchored proteins, which are indicative ofwhether cells are PNH Type III cells (little or no expression of GPI andGPI-anchored proteins), normal cells (Type I cells; having a relativelyhigh level of expression of GPI and GPI-anchored proteins as compared tothe Type III cells), and PNH Type II cells (having an intermediate levelof expression of GPI and GPI-anchored proteins as compared to Type Icells and Type III cells). The distinguishing or interrogating processcan involve, e.g., flow cytometry.

In some embodiments, the methods are used to detect the amount ofbinding of a reagent to RBCs from a patient sample. In some embodiments,the methods can be used to detect the amount of binding of a reagent towhite blood cells from a patient sample. White blood cells that areparticularly amenable to evaluation in the diagnostic methods describedherein include, e.g., granulocytes and monocytes (e.g., macrophages).

The samples can be from patients who have, are suspected of having, orat risk for developing paroxysmal nocturnal hemoglobinuria (PNH). Insome embodiments, the patients have one or more symptoms including,e.g., Coombs negative intravascular hemolysis, elevated LDH levels,recurrent iron deficiency anemia, thrombosis in unusual sites, episodicdysphagia, or abdominal pain.

In some embodiments, to aid in the identification of normal cells or PNHcells, a set of control cell populations can also be subjected to thedetection method. The control populations can be evaluated before,concurrently, or after the evaluation of the cell population ofinterest. As discussed in more detail below, a practitioner can chooseto subject a control population of cells known to be PNH Type II cells,a control population of cells known to be PNH Type III cells, and/or acontrol population of cells known to be normal or Type I cells to themethods to determine the typical amount, or average amount, of bindingof the reagent used to a particular type of cell. This controlinformation can be used to classify or identify cells (e.g., white bloodcells or red blood cells) of interest as normal cells, PNH Type IIcells, and/or PNH Type III cells.

Depending on the particular composition of the cell population withinthe biological sample, at least some cells of the population can bedistinguished from other cells based on a high amount of bound reagent,a low amount of bound reagent, or an intermediate level of boundreagent. In some cases, only cells with a high amount of bound reagentwill be present (for example, cells from a healthy patient or a patientwho does not have PNH). In some cases, a larger percentage of cells willhave little, or no, reagent bound to their surface (for example, whiteblood cells or RBCs from a PNH patient having a high percentage of PNHType III cells). In some embodiments, a population of cells contactedwith the reagent can be classified into high, low, and intermediatecategories based on the amount of reagent bound to the cells.

In some embodiments, one or more cells (e.g., one or more distinguishedor interrogated cells) can be classified based on the amount of reagentbound to their cell surface. As exemplified in the working examples anddepicted in FIG. 1, individual cells in a population can be readilyclassified as highly reagent bound, low or poorly reagent bound, andintermediately reagent bound using flow cytometry methods. For example,white blood cells obtained from a patient with PNH are contacted withtwo different reagents: a first reagent that binds to a GPI moiety(e.g., a detectably-labeled, non-lytic aerolysin protein) and a secondreagent that binds to a GPI-anchored protein (e.g., a detectably-labeledantibody that binds to human CD24). The first reagent and second reagentare labeled with different detectable labels. The contacted cells arethen subjected to flow cytometry. An artisan skilled in the art of flowcytometry would be readily able to use the methods to distinguishbetween cells based on the amount of binding of each reagent to thecells. See, e.g., Macey (2007) “Flow Cytometry: principles andapplications,” Humana Press (ISBN: 1588296911) and Brodsky et al. (2000)Am J Clin Pathol 114:459-466. As shown in FIG. 1, the flow cytometrymethods can readily be used to classify granulocytes obtained from a PNHpatient as having a high amount of binding of each of the reagents (cellpopulation at upper right; normal or Type I cells), a low amount ofbinding of each reagent (cell population at lower left; Type III cells),and an intermediate amount of binding of each reagent (cell populationat bottom center; Type II cells).

The classification of a cell can be performed by comparing the amount ofthe reagent bound to the cell to a control amount (e.g., a controlamount of binding of the reagent to PNH Type I cells, PNH Type II cells,and/or PNH Type III cells). The control amount of binding of the reagentto PNH Type I cells can be based on, e.g., the average amount ofobserved binding of the reagent to cells of the same histological typeobtained from one or more (e.g., two, three, four, five, six, seven,eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) healthy individuals.The control amount of binding of the reagent to PNH Type III cells canbe based on, e.g., the average amount of binding observed to cells ofthe same type obtained from one or more (e.g., two, three, four, five,six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) patientswith PNH. The control amount of binding of the reagent to PNH Type IIcells can be based on, e.g., the average amount of binding observed tocells of the same type obtained from one or more (e.g., two, three,four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 ormore) patients with PNH and having a detectable population of PNH TypeII cells of the same histological type (same lineage). For example, toclassify a white blood cell of interest based on the amount of reagentbound to the surface of the cell, a practitioner can compare the amountof reagent bound to the cell with the typical amount, or average amount,of reagent bound to a white blood cell known to be a PNH Type I whiteblood, a white blood cell known to be a PNH Type II white blood cell,and/or a white blood cell known to be a PNH Type III white blood cell.

In some embodiments, the distinguishing or classifying of a cell (e.g.,a white blood cell or RBC) of interest can be performed by determiningwhether the amount of a reagent bound to the cell falls within apredetermined range indicative of PNH Type I cells, PNH Type II cells,or PNH Type III cells of the same histological type. In someembodiments, the distinguishing or classifying of a hematopoietic cellof interest can include determining if the amount of reagent bound tothe surface of the cell falls above or below a predetermined cut-offvalue. A cut-off value is typically the amount of reagent bound to thesurface of a cell (or the amount of signal detected from a cell) aboveor below which is considered indicative of a certain class of cells,namely PNH Type I cells, PNH Type II cells, or PNH Type III cells.

Some cut-off values are not absolute in that diagnostic correlations(e.g., an amount of reagent bound to the surface of the cell andlikelihood that the cell is a PNH Type II cell) can still remainsignificant over a range of values on either side of the cutoff. It isunderstood that refinements in optimal cut-off values could bedetermined depending on the quality of reagents used, the sophisticationof statistical methods and detection device (e.g., flow cytometry) used,and on the number and source of samples interrogated. Therefore, cut-offvalues can be adjusted up or down, on the basis of periodicre-evaluations or changes in methodology or sample distribution.

As used herein, “thrombocytopenia” refers to a condition in which apatient has a platelet count of less than 200,000 (e.g., less than150,000; less than 140,000; less than 130,000; less than 120,000; lessthan 110,000; less than 100,000; or less than 90,000) platelets per μLof blood. In some embodiments, a patient with thrombocytopenia has aplatelet count of less than 100,000 platelets per μL of blood.

As described above, information related to the percentage of PNH Type IIcells can be used in methods for determining whether a patient is atincreased risk for developing thrombosis. The information related to thepercentage of PNH Type II cells (e.g., Type II white blood cells and/orType II red blood cells) in a biological sample from a patient can becommunicated (e.g., electronic or printed form) to a medicalpractitioner to be used by the practitioner for selecting an appropriatetherapeutic regimen for the patient. Based on a PNH Type II white bloodcell population of at least 1.2 (e.g., at least 1.2, 1.5, 2, 3, 5, 7, 9,10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47, 50, 52,55, 57, 60, 62, 65, or 65.3 or more) %, as compared to the total numberof white blood cells of the same histological type in the biologicalsample tested, the practitioner may determine that the patient is atrisk of developing thrombocytopenia, or may likely be thrombocytopenic.Likewise, a patient with a PNH Type II RBC population of at least 0.02(e.g., at least 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,71, 71.3, or 75 or more) % of the total RBC in the biological sampletested is much more likely to be thrombocytopenic than a patient whodoes not have a detectable PNH Type II RBC population or a patient witha PNH Type II RBC population lower than 0.02%. A patient with a PNH TypeII white blood cell population of at least 1.2% or a PNH Type II redblood cell population of at least 0.02% can be, e.g., at least 1.5(e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 11.5, 12, 12.5, 15, 20, 30, or even 40 or more) times aslikely to develop a thrombus than a normal individual or a patient thatdoes not have that percentage of PNH Type II cells.

The medical practitioner may request additional tests to determine theplatelet counts in the patient. Methods for determining platelet countsin a blood-derived sample from a subject are well known in the art ofmedicine and described in, e.g., Sallah et al. (1998) PostgraduateMedicine 103:209-210; Kottke-Marchant (1994) Hematol Oncol Clin NorthAm. 8:809-853; Redei et al. (1995) J Crit Illn 10:133-137; Butkiewicz etal. (2006) Thrombosis Research 118(2):199-204; Tomita et al. (2000) Am JHematol 63(3):131-135; and Schrezenmeier et al. (1998) Br J Haematol100(3):571-576.

If the patient is determined by the medical practitioner to bethrombocytopenic or to likely be thrombocytopenic, the practitioner mayselect, prescribe, or administer to the patient an anti-thrombocytopenictherapy. The anti-thrombocytopenic therapy can be, e.g., acorticosteroid, platelet transfusion, a splenectomy, or a plateletproduction-stimulating agent. The platelet production-stimulating agentcan be, e.g., thrombopoietin (TPO) or a thrombopoietin mimetic. See,e.g., Kuter and Begley (2002) Blood 100:3457-3469; Li et al. (2001)Blood 98:3241-3248; and Vadhan-Raj et al. (2000) Ann Intern Med132:364-368. A TPO mimetic peptide can have the amino acid sequencedepicted in FIG. 5 of U.S. Patent Application Publication No.20030049683, the disclosure of which (particularly FIG. 5) isincorporated by reference in its entirety.

If the percentage of PNH Type II white blood cells in a biologicalsample from a patient is about 1.2%, the medical practitioner may alsodetermine that the patient is at an increased risk for developingthrombosis. The medical practitioner may then select for the patient anappropriate anti-thrombotic therapy. For example, the practitioner mayselect, prescribe, or administer to the patient an anticoagulant orthrombolytic agent. The anticoagulant can be, e.g., coumadin, heparin,or derivatives thereof. The thrombolytic agent can be, e.g., a tissueplasminogen activator (e.g., Retavase™, Rapilysin™), streptokinase, or aurokinase-type plasminogen activator.

In some embodiments, a patient determined to have a PNH Type II whiteblood cell population of greater than or equal to 1.2% or a PNH Type IIred blood cell population of greater than or equal to 0.02% can bediagnosed as having PNH. In some embodiments, a patient diagnosed withhaving PNH or a previously diagnosed PNH patient who is determined tohave a PNH Type II white blood cell population greater than or equal to1.2% or a PNH Type II red blood cell population that is greater than orequal to 0.02% can be prescribed and/or treated with a complementinhibitor.

Any compounds which bind to or otherwise block the generation and/oractivity of any of the human complement components may be utilized inaccordance with the present disclosure. For example, an inhibitor ofcomplement can be, e.g., a small molecule, a nucleic acid or nucleicacid analog, a peptidomimetic, or a macromolecule that is not a nucleicacid or a protein. These agents include, but are not limited to, smallorganic molecules, RNA aptamers, L-RNA aptamers, Spiegelmers, antisensecompounds, double stranded RNA, small interfering RNA, locked nucleicacid inhibitors, and peptide nucleic acid inhibitors. In someembodiments, a complement inhibitor may be a protein or proteinfragment.

In some embodiments, antibodies specific to a human complement componentare useful herein. Some compounds include antibodies directed againstcomplement components C1, C2, C3, C4, C5 (or a fragment thereof; seebelow), C6, C7, C8, C9, Factor D, Factor B, Factor P, MBL, MASP-1, andMASP-2, thus preventing the generation of the anaphylatoxic activityassociated with C5a and/or preventing the assembly of the membraneattack complex associated with CSb.

Also useful in the present methods are naturally occurring or solubleforms of complement inhibitory compounds such as CR1, LEX-CR1, MCP, DAF,CD59, Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH.Other compounds which may be utilized to bind to or otherwise block thegeneration and/or activity of any of the human complement componentsinclude, but are not limited to, proteins, protein fragments, peptides,small molecules, RNA aptamers including ARC187 (which is commerciallyavailable from Archemix Corporation, Cambridge, Mass.), L-RNA aptamers,spiegelmers, antisense compounds, serine protease inhibitors, moleculeswhich may be utilized in RNA interference (RNAi) such as double strandedRNA including small interfering RNA (siRNA), locked nucleic acid (LNA)inhibitors, peptide nucleic acid (PNA) inhibitors, etc.

In some embodiments, the complement inhibitor inhibits the activation ofcomplement. For example, the complement inhibitor can bind to andinhibit the complement activation activity of C1 (e.g., C1q, C1r, orC1s) or the complement inhibitor can bind to and inhibit (e.g., inhibitcleavage of) C2, C3, or C4. In some embodiments, the inhibitor inhibitsformation or assembly of the C3 convertase and/or C5 convertase of thealternative and/or classical pathways of complement. In someembodiments, the complement inhibitor inhibits terminal complementformation, e.g., formation of the C5b-9 membrane attack complex. Forexample, an antibody complement inhibitor may include an anti-C5antibody. Such anti-C5 antibodies may directly interact with C5 and/orC5b, so as to inhibit the formation of and/or physiologic function ofC5b. Exemplary anti-C5 antibodies include, e.g., eculizumab (Soliris®;Alexion Pharmaceuticals, Inc., Cheshire, Conn.; see, e.g., Kaplan (2002)Curr Opin Investig Drugs 3(7):1017-23; Hill (2005) Clin Adv HematolOncol 3(11):849-50; and Rother et al. (2007) Nature Biotechnology25(11):1256-1488) and pexelizumab (Alexion Pharmaceuticals, Inc.,Cheshire, Conn.; see, e.g., Whiss (2002) Curr Opin Investig Drugs3(6):870-7; Patel et al. (2005) Drugs Today (Banc) 41(3):165-70; andThomas et al. (1996) Mol Immunol. 33(17-18):1389-401).

Methods for administering an appropriate anti-thrombotic therapy and/oran anti-thrombocytopenic therapy to a patient in need thereof are wellknown in the art of medicine.

In some embodiments, methods for determining whether a patient is at anincreased risk for developing thromobocytopenia or thrombosis can beaided by computer. For example, the methods can include receiving dataincluding a medical profile of a PNH patient, the profile comprisinginformation on the percentage of PNH Type II white blood cells of thetotal white blood cells of the same histological type (same lineage) ina biological sample from the patient; and processing at least theportion of the data containing the information to determine whether thepatient is at an increased risk for developing thrombosis. In anotherexample, the methods can include providing information on the percentageof PNH Type II white blood cells of the total white blood cells of thesame histological type in a biological sample from the patient;inputting the information into a computer; and calculating a parameterindicating whether the patient is at an increased risk for thrombosisusing the computer and the input information. The relative risk of thepatient for developing thrombocytopenia or thrombosis can be output bythe computer in print and/or can be stored on a computer-readablemedium.

Kits

Also featured herein are kits for use in: determining whether abiological sample from a patient contains a PNH Type II white blood cellpopulation and/or determining if a patient is at increased risk fordeveloping thrombocytopenia or thrombosis. The kits can include, e.g.,one or more of detectably-labeled conjugates selected from: an aerolysinconjugate (e.g., a non-lytic variant aerolysin protein conjugate) orconjugates of antibodies that bind to GPI-anchored proteins. The kitscan also include a control sample containing a GPI expressing cell orGPI bound particle; and optionally, instructions for detecting thepresence of a GPI expressing cell. The kits can also include one or moremeans for obtaining a biological sample (e.g., a blood sample) from ahuman and/or any of the kit components described above.

The following examples are intended to illustrate, not limit, theinvention.

EXAMPLES Example 1 Materials and Methods

A total of 2,921 patient peripheral blood samples were obtained to testfor the presence of PNH Type II cells and PNH Type III cells. The bloodsamples were drawn into sterile vials containing EDTA.

To determine the percentage of normal white blood cells (Type I cells),PNH Type II, and PNH Type III white blood cells present in each of thepatient samples, the peripheral blood was mixed and stained with one ormore of the following conjugates: a non-lytic aerolysin variant proteinconjugated to AlexaFluor® 488 (Protox Biotech FL2-S), an anti-CD24antibody conjugated to phycoerythrin (PE) (Beckman Coulter Clone ALB9),an anti-CD15 antibody conjugated to PC5 (Clone 80H5), and an anti-CD45antibody conjugated to PC7 (Clone J.33). After incubation of the bloodwith one or more of the above reagents for 15-30 minutes at roomtemperature, the blood was lysed with Immunoprep™ (Beckman Coulter) andwashed twice with PBA buffer (phosphate-buffered saline, 1% bovine serumalbumin, and 10 mM NaN₃). Cells are then re-suspended in PBA buffer andanalyzed using the FC 500 Flow Cytometer (Beckman Coulter). If a PNHType II or Type III granulocyte population was identified, the monocyteswere also interrogated for Type II or Type III population using patientblood contacted with one or more of the following conjugates: anon-lytic aerolysin variant protein conjugated to AlexaFluor® 488(Protox Biotech FL2-S), an antibody that binds to CD33 conjugated tophycoerythrin (PE) (Clone D3HL60.251), an antibody that binds to CD14conjugated to ECD (Clone RMO52), an antibody that binds to CD64conjugated to PC5 (Clone 22), and an antibody that binds to CD45conjugated to PC7 (Clone J.33), which allowed for lineage-specificgating on monocytes.

To determine the percentage of normal red blood cells (Type I cells),PNH Type II, and PNH Type III red blood cells 20 μl of peripheral bloodin EDTA was placed in 3 mL of phosphate buffered saline (PBS) and mixedthoroughly. 50 μl of diluted patient blood was contacted with one ormore of the following conjugates: an anti-CD235a antibody conjugated toFITC (Beckman Coulter clone 11E4B-7-6/KC16) and an anti-CD59 antibodyconjugated to PE (Invitrogen Clone MEM-43). The blood and conjugateswere incubated at room temperature in the dark for one hour whilevortexing every 15 minutes. After the one hour incubation, the blood waswashed twice with PBS, resuspended in PBS, and analyzed on an FC 500Flow Cytometer (Beckman Coulter).

Example 2

Whole blood samples from 2,921 patients, which samples were submittedfor diagnostic testing for PNH, were analyzed using a high-sensitivityflow cytometry-based assay to detect the expression level of GPI andGPI-anchored proteins on white blood cells (particularly granulocytes)to thereby determine the Type I, PNH Type II, and PNH Type III whiteblood cell (granulocyte) populations in each of the samples. The assaywas also used to detect the Type I, PNH Type II, and PNH Type III redblood cell populations in each of the patient samples. The methodsemployed a fluorescently-labeled non-lytic aerolysin protein variantalong with antibodies to specific GPI-anchored lineage-specific proteinantigens. An exemplary flow-cytometry analysis of one patient sample isdepicted in FIG. 1. Cells from a whole blood sample were contacted withthe fluorescently-labeled aerolysin reagent (Alexa Fluor) and aphycoeyrthrin (PE)-labeled antibody that binds to the GPI-anchoredprotein CD24. The cells of the whole blood sample were subjected to flowcytometry analysis and the granulocytes therein displayed based on theamount of signal detected from each reagent bound to the surface of thegranulocytes. As shown in FIG. 1, granulocytes with the highest amountof signal detected from the AlexaFluor and PE labels (upper right; TypeI cells) were separated from populations of granulocytes having a verylow or absent signal (lower left; Type III granulocytes) andgranulocytes producing an intermediate amount of signal (middlepopulation; Type II granulocytes).

The PNH red blood cell populations were interrogated using two reagents:a detectably-labeled antibody that binds to CD235 and adetectably-labeled reagent that binds to CD59. The PNH white blood cellpopulations were interrogated using the detectably-labeled aerolysinprotein and several antibodies to GPI-anchored lineage-specific cellsurface proteins including CD24, CD14, CD16, CD66b, and CD55.

216 of the patient samples had a detectable PNH Type III granulocytepopulation that was >0.01% of the total number of granulocytes in thesample and an absolute count of at least 50 PNH Type III granulocytes.Clinical information related to several parameters (e.g., hemoglobinlevels, LDH levels, and platelet counts) was available for 162 of thesepatients (see Table 1).

TABLE 1 Clinical Features of Patients with a Detectable PNH Type III orII granulocyte population (where clinical data were available.) CasesCases without with Type Type II II granulocytes granulocytes P-value (N= 19) (N = 143) Wilcoxin Median (range) Total PNH 87.20 11.40 <0.01granulocyte population (9.2-99.5) (0.01-99.9) (%) Median (range) PNHType 7.10 n/a n/a II granulocyte population (1.2-65.3) (%) Median(range) PNH Type 76.0 11.40 0.02 III granulocyte population (4.5-96.4)(0.01-99.9) (%) Median (range) PNH Type 3.30 0.20 <0.01 II RBCpopulation (%) (0.02-71.3)     (0-76.20) Median (range) PNH Type 16.102.90 0.01 III RBC population (%) (0.03-86.70)   (0-92.9) Median whiteblood cell 3.80 4.20 0.44 (×10⁹/L) Median absolute 2.07 2.15 0.70neutrophil count (cells/μL) Median RBC (×10¹²/L) 3.08 3.14 0.80 Medianhemoglobin (g/dL) 10.6 10.4 0.87 Median LDH (IU/L) 336 315 0.88 Medianplatelets (×10⁹/L) 54 116 0.01 Platelets <100 × 10⁹/L (%) 68.4 (13/19)44.0 (62/141) 0.05* *Fisher's exact test.Of the samples from patients in which clinical information wasavailable, 19 (8.8%) patient samples contained distinct Type IIgranulocyte populations, ranging from 1.2-65.3% of the total granulocytepopulation, with a median clone size of approximately 7%. In 4 of the 19patient samples, the Type II granulocyte population represented >50% ofthe total abnormal population (e.g., PNH Type II and Type III cells). In10 of the 19 patient samples, a PNH Type II monocyte population was alsodetected. An evaluation of the ability of various antibodies, specificfor individual GPI-linked proteins found on granulocytes, to detect PNHType II granulocytes indicated that the Type II granulocyte populationwas detectable in all cases using the detectably-labeled aerolysinreagent, but in decreasing percentages using antibodies specific forCD66b (88%), CD55 (50%), CD24 (47%), and CD16 (0%) (see Table 2). Theseresults indicate that the aerolysin-based conjugate is particularlyuseful to accurately detect PNH Type II granulocyte populations inpatient samples.

TABLE 2 Detection of Type II granulocytes using aerolysin or otheranti-GPI anchored-protein antibodies. Aerolysin CD24 CD66b CD55 CD1619/19 (100%) 9/19 (47%) 8/9 (88%) 4/8 (50%) 0/9 (0%)Patient samples containing PNH Type II granulocyte populations had asignificantly larger median total combined PNH Type II and PNH Type IIIgranulocyte population than those without Type II granulocytes (87%versus 11%; p=0.0003), as well as larger median Type II and Type III RBCpopulations, which reflects an increased ability of the method to detectPNH Type II white blood cell populations in patient samples with overalllarger PNH cell populations.

After comparison with the clinical data it was discovered that patientsamples with PNH Type II granulocyte populations also had lower medianplatelet (plt) counts (54×10⁹/L; p<0.01). See FIG. 2. Patient sampleswith PNH Type II granulocyte populations had similar peripheral whiteblood cell counts, peripheral red blood cell counts, absolute neutrophilcounts, and hemoglobin (Hgb) levels, compared to patient samples withoutdetectable Type II granulocyte populations (Table 1), indicating thatdifferences in platelet counts are likely not due to differences inunderlying bone marrow production. In other words, while the disclosureis in no way limited by any particular theory or mechanism of action, asPNH patients have dysregulated complement control due to the lack of theGPI-linked complement regulatory proteins CD55 and CD59, the decreasedplatelet counts observed in patients with detectable PNH Type IIgranulocyte clones may be due to increased terminal complement-mediatedplatelet consumption or destruction, which may in turn be associatedwith thrombosis, the leading cause of death among PNH patients.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

1. A method for predicting whether a patient is at an increased risk fordeveloping thrombosis, the method comprising: determining the percentageof PNH Type II white blood cells of the total white blood cells of thesame histological type in a biological sample from a patient; andpredicting whether the patient is at an increased risk for developingthrombosis, wherein the patient is at an increased risk for developingthrombosis if the percentage of PNH Type II white blood cells is greaterthan or equal to 1.2%.
 2. The method of claim 1, wherein the white bloodcells are i) granulocytes or ii) monocytes.
 3. (canceled)
 4. The methodof claim 1, wherein the biological sample is a whole blood sample. 5.(canceled)
 6. The method of claim 1, wherein a PNH Type II white bloodcell population that is between 1.2% to 65.3%, inclusive of 1.2% and65.3%, indicates that the patient is at an increased risk forthrombosis.
 7. The method of claim 1, wherein a PNH Type II white bloodcell population that is greater than or equal to a) 5%, b) 10%, c) 20%,or d) 50% indicates that the patient is at an increased risk forthrombosis. 8-10. (canceled)
 11. The method of claim 1, furthercomprising monitoring the patient for the development of at least onesymptom of thrombosis if the patient is at an increased risk ofdeveloping thrombosis.
 12. The method of claim 1, further comprisingselecting an anti-thrombotic therapy for the patient if the patient isat an increased risk of developing thrombosis.
 13. The method of claim12, wherein the anti-thrombotic therapy is an anticoagulant orthrombolytic agent.
 14. The method of claim 13, wherein theanticoagulant is coumadin, heparin, or derivatives thereof.
 15. Themethod of claim 13, wherein the thrombolytic agent is a tissueplasminogen activator, streptokinase, or a urokinase-type plasminogenactivator.
 16. The method of claim 1, further comprising administeringto the patient an anti-thrombotic therapy if the patient is at anincreased risk for developing thrombosis.
 17. A method for selecting atherapy for a patient, the method comprising: selecting one or both ofan anti-thrombotic therapy and an anti-thrombocytopenic therapy for apatient determined to have a PNH Type II white blood cell population ofgreater than or equal to 1.2%.
 18. A method for treating a patient, themethod comprising administering to a patient in need thereof one or bothof an anti-thrombotic therapy and an anti-thrombocytopenic therapy ifthe patient has a PNH Type II white blood cell population of greaterthan 1.2%.
 19. The method of claim 17 or 18, wherein the anti-thrombotictherapy is an anticoagulant or thrombolytic agent.
 20. The method ofclaim 17 or 18, wherein the anti-thrombocytopenic therapy is a platelettransfusion. 21-22. (canceled)
 23. The method of any one of claim 1, 17,or 18, wherein a non-lytic variant form of aerolysin protein is used todetermine the percentage of PNH Type II white blood cells.
 24. A methodfor identifying a PNH Type II white blood cell, the method comprising:contacting a plurality of white blood cells with a reagent that bindsto: (i) GPI or (ii) a GPI-anchored protein; and identifying one or moreof the white blood cells as PNH Type II white blood cells based on theamount of reagent bound to the cells, wherein an intermediate amount ofbinding of the reagent to a white blood cell, as compared to the amountof binding of the reagent to a PNH Type III white blood cell and theamount of binding of the reagent to a Type I white blood cell, indicatesthat the white blood cell is a PNH Type II white blood cell.
 25. Amethod for distinguishing between white blood cell populations, themethod comprising: contacting a plurality of white blood cells with areagent that binds to: (i) GPI or (ii) a GPI-anchored protein; anddistinguishing at least a portion of the white blood cells from otherwhite blood cells of the plurality based on the amount of reagent boundto the cells, wherein the PNH Type II white blood cells, if present, aresufficiently distinguished from the Type I white blood cells and PNHType III white blood cells of the same histological type to allow thepercentage of PNH Type II white blood cells of the total white bloodcells of the same histological type in the plurality to be determined.26. The method of claim 24 or 25, further comprising determining thepercentage of PNH Type II white blood cells of the total white bloodcells of the same histological type in the plurality.
 27. A method fordetermining the percentage of PNH Type II white blood cells, the methodcomprising: providing a plurality of white blood cells contacted with areagent that binds to: (i) GPI or (ii) a GPI-anchored protein;distinguishing at least a portion of white blood cells from other whiteblood cells of the plurality based on the amount of reagent bound to thecells, wherein the PNH Type II white blood cells, if present, aresufficiently distinguished from the Type I white blood cells and PNHType III white blood cells of the same histological type to allow thepercentage of PNH Type II white blood cells of the total white bloodcells of the same histological type in the plurality to be determined;and determining the percentage of PNH Type II white blood cells.
 28. Themethod of claim 26 or 27, further comprising determining the percentageof PNH Type III white blood cells.
 29. The method of claim 25 or 27,wherein the distinguishing comprises flow cytometry.
 30. The method ofany one of claims 24, 25, or 27, wherein the plurality of white bloodcells are obtained from a patient having, suspected of having, or atrisk of developing PNH. 31-32. (canceled)
 33. The method of anyone ofclaims 24, 25, or 27, wherein the reagent binds to a human GPI moiety.34. The method of claim 33, wherein the reagent comprises an aerolysinprotein.
 35. The method of claim 34, wherein the reagent comprises avariant form of aerolysin protein that is non-lytic or is substantiallynon-lytic as compared to the wild-type form of the protein.
 36. Themethod of claim 33, wherein the reagent comprises the amino acidsequence depicted in SEQ ID NO:2 or 7 wherein the threonine at position253 is substituted with a cysteine and the alanine at position 300 issubstituted for a cysteine.
 37. The method of claim 33, wherein thereagent is an antibody or an antigen-binding fragment thereof.
 38. Themethod of any one of claims 24, 25, or 27, wherein the reagent binds toa GPI-anchored protein.
 39. The method of claim 38, wherein theGPI-anchored protein is selected from the group consisting of alkalinephosphatase, 5′ nucleotidease acetylcholinesterase, dipeptidase, LFA-3,NCAM, PH-20, CD55, CD59, Thy-1, Qa-2, CD14, CD33, CD16 (the Fcγ receptorIII), carcinoembryonic antigen (CEA), CD24, CD66b, CD87, CD48, and CD52.40. The method of claim 38, wherein the reagent is an antibody or anantigen-binding fragment thereof.
 41. The method of any one of claims24, 25, or 27, wherein the plurality of white blood cells are contactedwith a reagent that bind to GPI and a reagent that binds to a GPI-linkedprotein.